CN101738372B - Optical detection device of liquid porphyrin array and method thereof for detecting amino acid - Google Patents
Optical detection device of liquid porphyrin array and method thereof for detecting amino acid Download PDFInfo
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Abstract
The invention discloses an optical detection device of a liquid porphyrin array and a method thereof for detecting amino acid, relating to a device and a method for optically detecting the amino acid by using porphyrin. The device mainly comprises a light source system, a reaction chamber, a spectrum collection system, a signal processing analysis and comparison system and a power system and is characterized in that the middle part of a substrate in the reaction chamber is divided into 10 blocks, 10 colorimetric wares are arranged, and liquid porphyrin detection solution is arranged in the 10colorimetric wares. In the method, the device is utilized to detect spectrograms of the amino acid in the liquid porphyrin detection solution by preparing porphyrin solution, amino acid solution, andthe liquid porphyrin detection solution, analyze and compare the spectrograms and judge the type of the amino acid. The invention has the advantages of simple method, convenient and rapid operation, low detection cost, favorable repeatability of detection, high accuracy, sensitivity and precision and non environmental pollution and can be widely applied to biological industry, medical industry, catalysis and material industry, biological pharmacy, food fermentation industry and brewing industry.
Description
Technical field
The invention belongs to amino acid detection technique field, be specifically related to amino acid whose device of porphyrin optical detection and detection method.
Background technology
Amino acid is to constitute biosome protein and with vital movement the most basic relevant material, is the base unit that constitutes protein molecule in vivo, with the vital movement of biology confidential relation arranged.It has special physiological function in antibody, be one of indispensable nutritional labeling in the biosome.Amino acid mainly contains following function: 1, constituting the base substance of human body, is the material base of life metabolism; 2, be nutrient in the food; 3, anti-ageing; 4, in the application of medical field.Amino acid pharmaceutically is being mainly used to prepare Hausmam Amin 20, also is used as medicine and is used for synthetic polypeptide medicaments.The compound preparation of being made up of several amino acids occupies important status in modern PN solution and " essential food " therapy, to keeping urgent patient's nutrition, rescue patient's life and have a positive effect, become one of medical kind indispensable in the modern medical service.Amino acid whose of a great variety, and effect separately also is not quite similar, and is used for the treatment of hepatopathy disease, disease of digestive tract, encephalopathic, cardiovascular disease, breathing problem separately and is used to improve muscle vitality, paediatrics nutrition and detoxifcation etc. as amino acid such as glutamic acid, arginine, asparatate, cystines.Hope has appearred in amino acid derivativges in treatment of cancer.Therefore, by amino acid is detected, can instruct clinical pathologic, physiologic research, medical diagnosis on disease, medicine is carried out toxicological study and safety evaluatio etc.The detection of amino acid metabolism situation will be applied to drug toxicology screening, to the research and development of medicine and safe handling etc., in scientific domains such as the early diagnosis of major disease, personalized treatment, traditional Chinese medicine combination, extremely extensive and important application prospects are arranged all.So amino acid whose check and analysis tool is played a very important role.
Have optical detection apparatus now and detect amino acid whose method, as application number is " essential amino acid determining instrument " patent of 89205054.3, and disclosed pick-up unit mainly is made up of photoelectric colorimetry light path system, photodetector system, A/D change-over circuit, special-purpose single-chip microprocessor system and power circuit etc.The major defect of this device is: 1. photodetector system is by photoelectric cell, zeroing, accent full scale resistance R 1, R2, R3, and switch, high precision, low drifting operating amplifier are formed, and therefore the structure more complicated makes detection speed relatively slow; What 2. special-purpose single-chip microprocessor system used is 8031 Single Chip Microcomputer (SCM) system, uses the program that need extend out and data space, pretty troublesome, make peripheral components many, and 8031 power consumption is also big, and performance is not high, uses limitedly, is not easy to popularization.3. can only measure the essential amino acid in native protein and the crude protein content." solid state optics detects amino acid (Solid-state optical detection of amino acids) " literary composition of 2003 the 91st volumes of and for example " sensor and driver B " (" Sensors and Actuators B ") 227-230 page or leaf, the amino acid whose method of disclosed detection is: Meso-four (4-sulfonic group phenyl) porphyrin (TPPS) individual layer is fixed on the cellulose membrane (being molecule-porous membrane tube), after hatching is spent the night and is cleaned, as the solid state optics sensor.In tested 4 kinds of L-amino acid (arginine, glycocoll, histidine and serine) solution, be under 7 conditions in the pH value, add sodium phosphate buffer agent respectively, prepare 4 tested seed amino acid buffer solution.4 tested seed amino acid buffer solution are positioned over respectively in the molecule-porous membrane tube of solid state optics sensor and react.Respectively will be not do not place cuvette, with the spectrum of Cary4E spectrophotometer and Grams/32 apparatus measures film and the spectrum change before and after the analytical reactions with the film of buffered with amino acid solution reaction with the film of Freamine reaction.The linear relationship of amino acid concentration point PSI-Plot in the cuvette of absorption spectrum correspondence.The shortcoming of this method is: (1) needs that porphyrin is carried out film and fixes, and it is clean to carrying out rinsing before and after the film forming to use several reagent, has increased operation and man-hour; (2) carry out the porphyrin film fixedly the time, needing hatching to spend the night, increasing the setup time of reaction detection greatly; (3) use filmogen and multiple buffer reagent, increased production cost; (4) cleaning fluid discharge is arranged, can bring certain environmental pollution.Roll up " array detection systems research of multimetering formula poison gas and spectral analysis " literary composition of the 1st phase " photoelectric project " January the 36th in 2009 for another example, disclosed array poison gas detection system, with the metalloporphyrin bar is sensitive materials, identification and trace concentration to the poison gas kind detect, and this system examines module, embedded hardware module and Data Management Analysis surely by optical detection module, poison gas stream module, gauge head precision mechanical transmission and the Control Software module is formed.The major defect of this system is: (1) needs earlier metalloporphyrin to be made air-sensitive reagent piece, then these reagent pieces are fixed on the glass-base, again organic protection layer and glass-base are glued together at last and be made into the air-sensitive array bar, therefore the operating process of making array is too complicated, consuming time longer, increased operation and man-hour; (2) when metalloporphyrin is made the air-sensitive array bar, this operating process has been used and many metalloporphyrin has been carried out immobilized material, has increased production cost greatly; (3) detected gas can only contact with the surface of metalloporphyrin air-sensitive array bar, and this has limited the abundant reaction of detected gas and metalloporphyrin to a certain extent.
Summary of the invention
The objective of the invention is to detect the weak point of method of amino-acids, a kind of optical detection device of liquid porphyrin array is provided and detects amino acid whose method at existing porphyrin.The present invention has easy and simple to handle, detects easily row, quick, and cost is low, energy efficient and resource, characteristics such as environmental nonpollution.
Principle of the present invention is: porphyrins is to be a kind of big ring macromolecular compound of parent with the porphines, and bigger plane macrocyclic structure is very capable with colour developing owing to having, and is a class ultra-high sensitive developer.Porphines is the big pi-conjugated system of 18 electronics that is formed by connecting by four methine bridged bonds by four pyrrole rings.Four meso-(5,10,15,20) on porphin ring position and position, eight β-(2,3,7,8,12,13,17,18) all can be replaced by other group and generate a series of porphine derivative, i.e. porphyrin.Each atom of porphyrin ring is in same plane substantially, forms a height conjugated system that has 11 conjugated double bonds that very easily is subjected to the electronic effect influence of pyrrole ring and methine.This architectural feature makes porphyrin both show the molecule distinguishability that rigid plane brings, and shows the abundant electronic spectrum that conjugated system causes again; The simultaneous altitude conjugated system makes that its chemical property is more stable, color is darker.Thereby porphyrin has numerous characteristics such as the chemical stability of hard and soft property, electronics resiliency, photoelectromagnetic, photosensitivity and height and spectral response be wide.Metalloporphyrin, because the formation of coordination bond makes the symmetry of porphyrin ring and axial coordination effect strengthen, binding site increases, the more nonmetal porphyrin of character is more active.Liquid porphyrin is compared with solid-state porphyrin, and preparation is simple, and can fully react easier image data with Freamine.Single sulfonic group phenyl porphyrin is because the access of sulfonic acid group makes the avtive spot of porphyrin increase, and it is excellent more to detect effect.In liquid porphyrin array, utilization has the porphyrin and the different aminoacids solution reaction of different substituents, by spectra collection system---fiber spectrometer, gather and analyze the spectrum after the amino acid molecular interphase interaction in porphyrin and the different aminoacids solution, foundation is based on the amino acid detection method of molecular level, thereby the realization liquid porphyrin array is to the optical detection of amino acid molecular identification.
Realize that the object of the invention technical scheme is: a kind of optical detection device of liquid porphyrin array mainly comprises: light-source system, reaction chamber, spectra collection system, signal processing analysis comparison system and power-supply system.Light-source system mainly is made up of visible light source (being iodine-tungsten lamp), lens, fibre bundle (i.e. 10 incident opticals).Reaction chamber mainly is made up of two transparent glass, reactive tank, cuvette and substrates.The spectra collection system mainly is made up of transmitted light receiver, optical fiber, micro spectrometer, leading screw and stepper motor and Drive and Control Circuit.The spectra collection system detects one by one to the tested amino acid whose spectroscopic data in the cuvette in the reactive tank of reaction chamber.The signal processing analysis comparison system is an embedded OS, tested amino acid whose spectroscopic data in the reactive tank that main receiving spectrum acquisition system is gathered one by one in the cuvette, the row mode of going forward side by side identification and Treatment Analysis relatively reach data and preserve, print and output, and by drive control circuit stepper motor and then control spectra collection system.Power-supply system provides power supply to micro spectrometer and signal processing analysis comparison system and Drive and Control Circuit respectively.Feature is: the middle part of the inherent substrate of reaction chamber is divided into 10, at the middle part of every substrate one reactive tank is set, each reactive tank is rectangular glass case, its length is that 7～105mm, width are 12～18mm, highly are 42～66mm, in 10 cuvettes in 10 reactive tanks, place liquid porphyrin respectively and detect solution, be built into the liquid porphyrin detection arrays.It is in the liquid porphyrins such as single sulfonic group phenyl porphyrin, zinc protoporphyrin, iron porphyrin, manganoporphyrin, nickel-porphyrin, cobalt porphyrin, copper porphyrin, plumbous porphyrin, platinum porphyrins, rhodium porphyrin 1～10 kind mixed liquor with tested Freamine that its liquid porphyrin detects solution, and its liquid porphyrin: the volume ratio of tested Freamine is 1: 0.7～1.5.The concrete liquid porphyrin of placing detects solution in the cuvette, determines according to detecting amino acid whose needs.The signal processing analysis comparison system can carry out pattern-recognition and Treatment Analysis to detecting data, can also preserve data, and can select and carry out comparison, judgement between data automatically.
The amino acid whose method of a kind of liquid porphyrin array optical detection, utilize described optical detection device of liquid porphyrin array, through preparation porphyrin solution and Freamine and liquid porphyrin detection solution, measure liquid porphyrin and detect amino acid whose spectrogram in the solution, then spectrogram is compared to analyze and judge amino acid whose kind.Its concrete grammar step is as follows:
(1) measures amino acid whose basic spectrogram
1) preparation porphyrin solution
With porphyrin (is single sulfonic group phenyl porphyrin, zinc protoporphyrin, iron porphyrin, manganoporphyrin, nickel-porphyrin, the cobalt porphyrin, copper porphyrin, plumbous porphyrin, platinum porphyrins, in the rhodium porphyrin etc. 10 kinds), be the photaesthesia agent, with organic solvent (is absolute ethyl alcohol or chloroform or chlorobenzene or N, dinethylformamide etc.) be the constant volume agent, quality in porphyrin: the volume of organic solvent ratio is 1: 12.5～50 ratio, respectively 10 kinds of porphyrins are added in the organic solvent, after the stirring and dissolving, the porphyrin solution that just to prepare 10 kinds of porphyrin mass concentrations respectively be 0.02～0.08g/L, be positioned in the refrigerator respectively, and under 3～4 ℃, keep in Dark Place, standby.
2) preparation known amino acid solution
The (1)-1) step finish after, respectively according to a kind quality in 10 kinds of the known amino acids (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine etc.): the volume ratio of deionized water is 1: 17.9～31.25 ratio, in 10 kinds of known amino acids, add deionized water respectively, after stirring, just prepare 10 kinds of known amino acid solution respectively.
3) the preparation liquid porphyrin detects solution
The (1)-2) step finish after, respectively according to the (1)-1) 10 kinds of porphyrin liquor capacities preparing of step: the (1)-2) volume ratio of 10 kinds of known amino acid solution preparing of step is 1: 0.7～1.5 ratio, in 10 kinds of porphyrin solution, add a kind of known amino acid solution respectively, after mixing, just prepare 10 kinds of liquid porphyrins respectively and detect solution, until in 10 kinds of porphyrin solution, added respectively till 10 kinds of known amino acid solution, just prepared 100 kinds of liquid porphyrins and detect solution.
4) the basic spectrogram of measurement known amino acid
The (1)-3) step finish after, at every turn with the (1)-3) 10 kinds of liquid porphyrins in the step in prepare 100 kinds detect liquid, be positioned over respectively in 10 cuvettes in the reactive tank of described optical detection device of liquid porphyrin array, be built into the liquid porphyrin detection arrays.Then, open the light source and the energized of described optical detection device of liquid porphyrin array, the spectra collection system of described optical detection device of liquid porphyrin array detects the known amino acid that the liquid porphyrin in 10 cuvettes detects in the solution, record the basic spectrogram of 10 kinds of known amino acids in 10 cuvettes, and respectively its basic spectrogram and data are preserved and printout by the signal processing analysis comparison system, until measuring the (1)-3) 100 kinds of liquid porphyrins preparing of step detect till the basic spectrogram of known amino acid of solution.
(2) detect amino acid
1) prepares tested Freamine
(1) step finish after, according to detecting amino acid whose requirement, with tested amino acid (being 1～10 kind in glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, the lysine etc.), respectively according to tested amino acid masses: the volume ratio of deionized water is 1: 17.9～31.25 ratio, in 1～10 kind of tested amino acid, add deionized water respectively, after stirring, just prepare 1～10 kind of tested Freamine.
2) the preparation liquid porphyrin detects solution
The (2)-1) step finish after, according to detecting requirement, in the (1)-1) the porphyrin liquor capacity prepared of step: the (2)-1) volume ratio of the tested Freamine prepared of step is 1: 0.7～1.5 ratio, respectively the (1)-1) add the (2)-1 in 10 kinds of porphyrin solution preparing of step) 1～10 kind of tested Freamine preparing of step, after mixing, just prepare corresponding liquid porphyrin and detect solution.
3) measure tested amino acid whose spectrogram
The (2)-2) after the step finishes, earlier according to detecting requirement, with the (2)-2) liquid porphyrin prepared of step detects in 10 cuvettes in the reactive tank that solution is positioned over described optical detection device of liquid porphyrin array, is built into liquid porphyrin array.Then, open the light source and the energized of described optical detection device of liquid porphyrin array again, the spectra collection system of described optical detection device of liquid porphyrin array detects the tested amino acid that the liquid porphyrin in 10 cuvettes detects in the solution, records 10 kinds of tested amino acid whose spectrograms in 10 cuvettes.And the signal processing analysis comparison system by described optical detection device of liquid porphyrin array, automatically its spectrogram and data are preserved and printout.
(3) analysis relatively reaches judgement
(2) step finish after, the signal processing analysis comparison system of described optical detection device of liquid porphyrin array, automatically with the (2)-3) the tested amino acid whose spectrogram that gets of pacing, with preserve in the native system the (1)-4) after the basic spectrogram of the known amino acid that gets of pacing analyzes relatively, judge tested amino acid whose kind (being which kind of amino acid), and the result is preserved and printout.And manually with the (2)-3) the tested amino acid whose spectrogram and the (1)-4 of step printout) after the basic spectrogram that goes on foot printout analyzes relatively, also judge tested amino acid whose kind (promptly carrying out artificial nuclear), thereby accurately detect tested amino acid.
After the present invention adopted technique scheme, the main effect of the present invention was:
(1) the inventive method has realized that liquid porphyrin is to amino acid whose detection.Adopt the inventive method to detect amino acid, have fabulous repeatability and stable, the accuracy height has guaranteed the reliable and stable of quantitative and The qualitative analysis.The amino acid detectability reaches 10
-5Mol～10
-7The mol order of magnitude, the sensitivity of detection is good, precision is high, has satisfied the requirement of trace analysis.So it is a kind of more excellent method that the present invention detects amino acid with liquid porphyrin.
(2) the inventive method detection amino acid process is simple, the time spent is shorter.Amino acid whose check and analysis process is finished within 30min, has greatly improved work efficiency.
(3) detect amino acid with liquid porphyrin, and consumption is few, solution system only needs water and organic solvent to make solvent, does not need special reagents such as buffer solution, makes that the detection cost is very low, again non-environmental-pollution.
(4) adopt the inventive method to detect amino acid, in preparation process simple, the non-environmental-pollution of method, economize on resources, cost is lower.The inventive method is simple, need not add filmogen, has reduced many processing steps.In testing process, do not need complicated instrument and equipment, adopt fiber spectrometer to detect, avirulence, easy and simple to handle, the liquid array stability of the porphyrin that makes is good, continuous detecting when can be used for a plurality of parallel samples of a seed amino acid or different seed amino acid.
(5) easy to utilize.Because the inventive method is simple, easy and simple to handle, only existing array optical pick-up unit is just improved a little and can detect, and gained detects the spectrum good reproducibility, stability is high, detection time is short, precision is high.Thereby easy to utilize, even has the feasibility that industrialization is produced.
Adopt the inventive method, can be widely used in having a good application prospect and practical value in the fields such as biology, medical science, catalysis and material, bio-pharmaceuticals, food fermentation and brewery industry.
Description of drawings
Fig. 1 is the fiber spectrometer structural representation of optical detection device of liquid porphyrin array;
Among the figure: 1 light source, 2 lens, 3 fibre bundles, 4,7 transparent glass, 5 reactive tanks, 6 substrates, 8 transmitted light receivers, 9 optical fiber, 10 leading screws, 11 stepper motors, 12 micro spectrometers, 13 signal processing analysis comparison systems, 14 Drive and Control Circuit, 15 power-supply systems.
Fig. 2 is single sulfonic group phenyl porphyrin (TPPS in the embodiment 1
1) detect the spectrogram of the wavelength-absorbance of 10 kinds of unknown amino acids;
Fig. 3 is the enlarged drawing of Fig. 2 medium wave peak part.
Curve among Fig. 2,3: be followed successively by TPPS from top to bottom respectively
1Detect the characteristic light spectrogram at the maximum absorbance place of glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
A kind of optical detection device of liquid porphyrin array mainly comprises: light-source system, reaction chamber, spectra collection system, signal processing analysis comparison system 13 and power-supply system 15.Light-source system mainly is made up of visible light source 1 (being iodine-tungsten lamp), lens 2, fibre bundle 3 (i.e. 10 incident opticals).Reaction chamber mainly is made up of two transparent glass 4,7, reactive tank 5, cuvette and substrate 6.The spectra collection system mainly is made up of transmitted light receiver 8, optical fiber 9, micro spectrometer 12, leading screw 10 and stepper motor 11 and Drive and Control Circuit 14.Signal processing analysis comparison system 13 is an embedded OS, tested amino acid whose spectroscopic data in the reactive tank 5 that main receiving spectrum acquisition system is gathered one by one in the cuvette, the row mode of going forward side by side identification and Treatment Analysis relatively reach data and preserve, print and output, and by Drive and Control Circuit 14 control step motors 11 and then control spectra collection system.Power-supply system 15 provides power supply to micro spectrometer 12 and signal processing analysis comparison system 13 and Drive and Control Circuit 14 respectively.Feature is: the middle part of the inherent substrate 6 of reaction chamber is divided into 10, one reactive tank 5 is set at the middle part of every substrate 6, each reactive tank 5 is rectangular glass case, its length be 7mm, width be 12mm, highly for 42mm, in 10 cuvettes in 10 reactive tanks 5, place liquid porphyrin respectively and detect solution, be built into the liquid porphyrin detection arrays.
The concrete implementation step of the amino acid whose method of a kind of liquid porphyrin array optical detection is as follows:
(1) measures amino acid whose basic spectrogram
1) preparation porphyrin solution
With single sulfonic group phenyl porphyrin is the photaesthesia agent, with the absolute ethyl alcohol organic solvent is the constant volume agent, quality in single sulfonic group phenyl porphyrin: absolute ethyl alcohol volume of organic solvent ratio is 1: 13.5 a ratio, single sulfonic group phenyl porphyrin is added in the absolute ethyl alcohol organic solvent, after the stirring and dissolving, just preparing mass concentration is single sulfonic group phenyl porphyrin solution of 0.074g/L, is positioned in the refrigerator, under 3 ℃, keep in Dark Place, standby.
2) preparation known amino acid solution
The (1)-1) step finish after, according to a kind quality in 10 kinds of the known amino acids (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 17.9 a ratio, in 10 kinds of known amino acids, add deionized water respectively, after stirring, just prepare 10 kinds of known amino acid solution respectively.
3) the preparation liquid porphyrin detects solution
The (1)-2) step finish after, respectively according to the (1)-1) single sulfonic group phenyl porphyrin liquor capacity of preparing of step: the (1)-2) volume ratio of a kind of known amino acid solution in step prepare 10 kinds is 1: 1.5 a ratio, the (1)-2) add the (1)-1 respectively in 10 kinds of known amino acid solution preparing of step) single sulfonic group phenyl porphyrin solution of preparing of step, after mixing, just prepare 10 kinds of single sulfonic group phenyl porphyrins respectively and detect solution.
4) the basic spectrogram of measurement known amino acid
The (1)-3) after the step finishes, with the (1)-3) prepare 10 kinds single sulfonic group phenyl porphyrins detect solution in the step, are positioned over respectively in 10 cuvettes in the reactive tank 5 of described optical detection device of liquid porphyrin array, are built into the liquid porphyrin detection arrays.Then, open the light source 1 and the energized of described optical detection device of liquid porphyrin array, the spectra collection system of described optical detection device of liquid porphyrin array detects the known amino acid that the single sulfonic group phenyl porphyrin in 10 cuvettes detects in the solution.Record the basic spectrogram of 10 kinds of known amino acids in 10 cuvettes, and respectively its spectrogram and data are preserved and printout by signal processing analysis comparison system 13.
(2) detect amino acid
1) prepares tested Freamine
(1) step finish after, according to detecting amino acid whose requirement, according to a kind quality in 10 kinds in the tested amino acid (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 17.9 a ratio, in 10 kinds of tested amino acid, add deionized water respectively, after stirring, just prepare 10 kinds of tested Freamines.
2) the preparation liquid porphyrin detects solution
The (2)-1) step finish after, according to detecting requirement, in the (1)-1) single sulfonic group phenyl porphyrin liquor capacity of preparing of step: the (2)-1) volume ratio of a kind of tested Freamine in step prepare 10 kinds is 1: 1.5 a ratio, respectively the (2)-1) add the (1)-1 in 10 kinds of tested Freamines preparing of step) single sulfonic group phenyl porphyrin solution of preparing of step, after mixing, just prepare 10 kinds of tested single sulfonic group phenyl porphyrins and detect solution.
3) measure tested amino acid whose spectrogram
The (2)-1) step finish after, earlier according to detecting requirement, with the (2)-2) step prepare 10 kinds tested single sulfonic group phenyl porphyrins detect in 10 cuvettes in the reactive tank 5 that solution are positioned over described optical detection device of liquid porphyrin array, are built into liquid single sulfonic group phenyl porphyrin array.Then, open the light source 1 and the energized of described optical detection device of liquid porphyrin array again, the spectra collection system of described optical detection device of liquid porphyrin array detects the tested amino acid that the tested single sulfonic group phenyl porphyrin in 10 cuvettes detects in the solution, records 10 kinds of tested amino acid whose spectrograms in 10 cuvettes.And the signal processing analysis comparison system 13 by described optical detection device of liquid porphyrin array, automatically its spectrogram and data are preserved and printout.
(3) analysis relatively reaches judgement
(2) step finish after, the signal processing analysis comparison system 13 of described optical detection device of liquid porphyrin array, automatically with the (2)-3) the tested amino acid whose spectrogram that gets of pacing, with preserve in the native system the (1)-4) after the basic spectrogram of 10 kinds of known amino acids getting of pacing analyzes relatively, judge tested amino acid whose kind (being which kind of amino acid), and the result is preserved and printout.And manually with the (2)-3) the tested amino acid whose spectrogram and the (1)-4 of step printout) after the basic spectrogram that goes on foot printout analyzes relatively, also judge tested amino acid whose kind (promptly carrying out artificial nuclear), thereby accurately detect tested amino acid.
Embodiment 2
A kind of optical detection device of liquid porphyrin array, with embodiment 1, wherein: the length of each reactive tank 5 be 52.4mm, width be 15mm, highly for 45.5mm.
The concrete implementation step of the amino acid whose method of a kind of liquid porphyrin array optical detection, with embodiment 1, wherein:
The (1)-1) in the step: with the zinc protoporphyrin is the photaesthesia agent, with the chloroform organic solvent is the constant volume agent, and by the quality of zinc protoporphyrin: chloroform volume of organic solvent ratio is 1: 12.5, and preparing mass concentration is the zinc protoporphyrin solution of 0.08g/L, under 4 ℃, keep in Dark Place, standby.
The (1)-2) in the step: a kind quality in 10 kinds of the known amino acids (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 31.25, prepares 10 kinds of known amino acid solution.
The (1)-3) in the step: the (1)-1) go on foot the zinc protoporphyrin liquor capacity of preparing: the volume ratio that the (1)-2) goes on foot a kind of known amino acid solution in 10 kinds that prepare is 1: 0.7.
The (2)-1) in the step: a kind quality in 10 kinds in the tested amino acid (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 31.25, prepares 10 kinds of tested Freamines.
The (2)-2) in the step: the (1)-1) go on foot the zinc protoporphyrin liquor capacity of preparing: the volume ratio that the (2)-1) goes on foot a kind of tested Freamine in 10 kinds that prepare is 1: 0.7.
Embodiment 3
A kind of optical detection device of liquid porphyrin array, with embodiment 1, wherein: the length of each reactive tank 5 be 105mm, width be 18mm, highly for 66mm.
The concrete implementation step of the amino acid whose method of a kind of liquid porphyrin array optical detection, with embodiment 1, wherein:
The (1)-1) in the step: with the cobalt porphyrin is the photaesthesia agent, with the chlorobenzene organic solvent is the constant volume agent, and by the quality of cobalt porphyrin: chlorobenzene volume of organic solvent ratio is 1: 40, and preparing mass concentration is the cobalt porphyrin solution of 0.025g/L, under 3 ℃, keep in Dark Place, standby.
The (1)-2) in the step: a kind quality in 10 kinds of the known amino acids (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 24.6, prepares 10 kinds of known amino acid solution.
The (1)-3) in the step: the (1)-1) go on foot the cobalt porphyrin liquor capacity of preparing: the volume ratio that the (1)-2) goes on foot a kind of known amino acid solution in 10 kinds that prepare is 1: 1.5.
The (2)-1) in the step: a kind quality in 10 kinds in the tested amino acid (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 24.6, prepares 10 kinds of tested Freamines.
The (2)-2) in the step: the (1)-1) go on foot the cobalt porphyrin liquor capacity of preparing: the volume ratio that the (2)-1) goes on foot a kind of tested Freamine in 10 kinds that prepare is 1: 1.5.
Embodiment 4
A kind of optical detection device of liquid porphyrin array, with embodiment 1, wherein: the length of each reactive tank 5 be 52.4mm, width be 12.4mm, highly for 45mm.
The concrete implementation step of the amino acid whose method of a kind of liquid porphyrin array optical detection, with embodiment 1, wherein:
The (1)-1) in the step: with the iron porphyrin is the photaesthesia agent, with N, the dinethylformamide organic solvent is the constant volume agent, press the quality of iron porphyrin: N, dinethylformamide volume of organic solvent ratio is 1: 50, preparing mass concentration is the iron porphyrin solution of 0.02g/L, keeps in Dark Place under 4 ℃, standby.
The (1)-2) in the step: a kind quality in 10 kinds of the known amino acids (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 17.9, prepares 10 kinds of known amino acid solution.
The (1)-3) in the step: the (1)-1) go on foot the iron porphyrin liquor capacity of preparing: the volume ratio that the (1)-2) goes on foot a kind of known amino acid solution in 10 kinds that prepare is 1: 0.7.
The (2)-1) in the step: a kind quality in 10 kinds in the tested amino acid (being glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine): the volume ratio of deionized water is 1: 17.9, prepares 10 kinds of tested Freamines.
The (2)-2) in the step: the (1)-1) go on foot the iron porphyrin liquor capacity of preparing: the volume ratio that the (2)-1) goes on foot a kind of tested Freamine in 10 kinds that prepare is 1: 0.7.
A kind of optical detection device of liquid porphyrin array, with embodiment 1, wherein: the length of each reactive tank 5 be 18.5mm, width be 23mm, highly for 100mm.
The concrete implementation step of the amino acid whose method of a kind of liquid porphyrin array optical detection, with embodiment 1, wherein:
The (1)-1) in the step: with 10 kinds of porphyrins (being single sulfonic group phenyl porphyrin, zinc protoporphyrin, iron porphyrin, manganoporphyrin, nickel-porphyrin, cobalt porphyrin, copper porphyrin, plumbous porphyrin, platinum porphyrins, rhodium porphyrin), be the photaesthesia agent, with the chloroform organic solvent is the constant volume agent, quality by a kind of porphyrin in 10 kinds of porphyrins: chloroform volume of organic solvent ratio is 1: 12.5, the porphyrin solution that to prepare 10 kinds of porphyrin mass concentrations be 0.08g/L, under 4 ℃, keep in Dark Place, standby.
The (1)-2) in the step: the quality of known glycocoll: the volume ratio of deionized water is 1: 17.9, prepares known glycine solution.
The (1)-3) in the step: the (1)-1) a kind of porphyrin liquor capacity in 10 kinds of porphyrins preparing of step: the (1)-2) volume ratio of the known glycine solution prepared of step is 1: 0.7.
The (2)-1) in the step: the quality of tested glycocoll: the volume ratio of deionized water is 1: 17.9, prepares tested glycine solution.
The (2)-2) in the step: the (1)-1) a kind of porphyrin liquor capacity in 10 kinds of porphyrins preparing of step: the (2)-1) volume ratio of the tested glycine solution prepared of step is 1: 0.7.
A kind of optical detection device of liquid porphyrin array with embodiment 1, wherein is: the length of each reactive tank 5 be 9mm, width be 17.5mm, highly for 65mm.
The concrete implementation step of the amino acid whose method of a kind of liquid porphyrin array optical detection, with embodiment 1, wherein:
The (1)-1) in the step: with 5 kinds of porphyrins (being single sulfonic group phenyl porphyrin, zinc protoporphyrin, iron porphyrin, manganoporphyrin, cobalt porphyrin), be the photaesthesia agent, with the chloroform organic solvent is the constant volume agent, quality by a kind of porphyrin in 5 kinds of porphyrins: chloroform volume of organic solvent ratio is 1: 12.5, the porphyrin solution that to prepare 5 kinds of porphyrin mass concentrations be 0.08g/L, under 4 ℃, keep in Dark Place, standby.
The (1)-2) in the step: a kind quality in 5 kinds of the known amino acids (being glycocoll, methionine, glutamine, glutamic acid, histidine): the volume ratio of deionized water is 1: 12.5, prepares 5 kinds of known amino acid solution.
The (1)-3) in the step: the (1)-1) a kind of porphyrin liquor capacity in 5 kinds of porphyrins preparing of step: the (1)-2) volume ratio of a kind of known amino acid solution in step prepare 5 kinds is 1: 0.7.
The (2)-1) in the step: a kind quality in 5 kinds in the tested amino acid (being glycocoll, methionine, glutamine, glutamic acid, histidine): the volume ratio of deionized water is 1: 12.5, prepares 5 kinds of tested Freamines.
The (2)-2) in the step: the (1)-1) a kind of porphyrin liquor capacity in 5 kinds of porphyrins preparing of step: the (2)-1) volume ratio of a kind of tested Freamine in step prepare 5 kinds is 1: 0.7.
Experimental result
With embodiment 1 10 seed amino acids (glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, lysine etc.) are detected, draw the detection spectrogram, wherein single sulfonic group phenyl porphyrin (TPPS
1) to the detection spectrogram of 10 seed amino acids as shown in Figure 2, TPPS
1Near the characteristic peak of detection response results 415nm to 10 seed amino acids amplifies spectrogram as shown in Figure 3.
Interpretation:
Find out TPPS from Fig. 2 and Fig. 3
1The ultraviolet-visible light spectrum that 10 seed amino acids are detected has remarkable difference, 10 seed amino acids can be distinguished according near the characteristic peak intensity the 415nm.
Detection solution in the cuvette in other nine reactive tanks can be distinguished preferably to 10 seed amino acids equally, with 10 kinds detect solution to the detection spectrogram of same seed amino acid as one group of data, these group data just have uniqueness, thereby can be used as this amino acid whose detection spectrogram.
In sum, the inventive method can realize amino acid whose detection, and repeatability that detects and good stability, accuracy height, the sensitivity that detects is good, precision is high, and the present invention has easy and simple to handlely, detects easily row, fast, cost is low, energy efficient and resource, characteristics such as environmental nonpollution are with a wide range of applications at aspects such as the particularly unicellular diagnosis of cell, pathology, pharmacology, genomic medicine screening, medicine equipments.
Claims (1)
1. amino acid whose method of liquid porphyrin array optical detection, utilize optical detection device of liquid porphyrin array, this device mainly comprises: light-source system, reaction chamber, the spectra collection system, signal processing analysis comparison system (13) and power-supply system (15), the middle part that it is characterized in that the inherent substrate of reaction chamber (6) is divided into 10, at the middle part of every substrate (6) reactive tank (5) is set, each reactive tank (5) is rectangular glass case, its length is 7～105mm, width is 12～18mm, highly be 42～66mm, in 10 cuvettes in 10 reactive tanks (5), place liquid porphyrin respectively and detect solution, it is characterized in that concrete method step is as follows:
(1) measures amino acid whose basic spectrogram
1) preparation porphyrin solution
With porphyrin, it is single sulfonic group phenyl porphyrin, zinc protoporphyrin, iron porphyrin, manganoporphyrin, nickel-porphyrin, the cobalt porphyrin, copper porphyrin, plumbous porphyrin, platinum porphyrins, in the rhodium porphyrin 10 kinds, be the photaesthesia agent, with organic solvent, be absolute ethyl alcohol or chloroform or chlorobenzene or N, dinethylformamide is the constant volume agent, quality in porphyrin: the volume of organic solvent ratio is 1: 12.5～50 ratio, respectively 10 kinds of porphyrins are added in the organic solvent, after the stirring and dissolving, the porphyrin solution that just to prepare 10 kinds of porphyrin mass concentrations respectively be 0.02～0.08g/L is positioned in the refrigerator respectively, and keeps in Dark Place under 3～4 ℃;
2) preparation known amino acid solution
The (1)-1) step finish after, respectively according to known amino acid, be a kind quality in glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, the lysine: the volume ratio of deionized water is 1: 17.9～31.25 ratio, in 10 kinds of known amino acids, add deionized water respectively, stir;
3) the preparation liquid porphyrin detects solution
The (1)-2) step finish after, respectively according to the (1)-1) 10 kinds of porphyrin liquor capacities preparing of step: the (1)-2) volume ratio of 10 kinds of known amino acid solution preparing of step is 1: 0.7～1.5 ratio, in 10 kinds of porphyrin solution, add a kind of known amino acid solution respectively, mix, until in 10 kinds of porphyrin solution, added respectively till 10 kinds of known amino acid solution;
4) the basic spectrogram of measurement known amino acid
The (1)-3) step finish after, at every turn with the (1)-3) 10 kinds of liquid porphyrins in the step in prepare 100 kinds detect liquid, be positioned over respectively in 10 cuvettes in the reactive tank (5) of described optical detection device of liquid porphyrin array, then, open the light source (1) and the energized of described optical detection device of liquid porphyrin array, the spectra collection system of described optical detection device of liquid porphyrin array detects the known amino acid that the liquid porphyrin in 10 cuvettes detects in the solution, record the basic spectrogram of 10 kinds of known amino acids in 10 cuvettes, and respectively its basic spectrogram and data are preserved and printout by signal processing analysis comparison system (13), until measuring the (1)-3) 100 kinds of liquid porphyrins preparing of step detect till the basic spectrogram of known amino acid of solution;
(2) detect amino acid
1) prepares tested Freamine
(1) step finish after, according to detecting amino acid whose requirement, with tested amino acid, it is 1～10 kind in glycocoll, methionine, valine, proline, serine, tyrosine, glutamine, glutamic acid, histidine, the lysine, respectively according to tested amino acid masses: the volume ratio of deionized water is 1: 17.9～31.25 ratio, in 1～10 kind of tested amino acid, add deionized water respectively, stir;
2) the preparation liquid porphyrin detects solution
The (2)-1) step finish after, according to detecting requirement, in the (1)-1) the porphyrin liquor capacity prepared of step: the (2)-1) volume ratio of the tested Freamine prepared of step is 1: 0.7～1.5 ratio, respectively the (1)-1) add the (2)-1 in 10 kinds of porphyrin solution preparing of step) 1～10 kind of tested Freamine preparing of step, mix;
3) measure tested amino acid whose spectrogram
The (2)-2) step finish after, earlier according to detecting requirement, with the (2)-2) liquid porphyrin prepared of step detects in 10 cuvettes in the reactive tank (5) that solution is positioned over described optical detection device of liquid porphyrin array, then, open the light source (1) and the energized of described optical detection device of liquid porphyrin array again, the spectra collection system of described optical detection device of liquid porphyrin array detects the tested amino acid that the liquid porphyrin in 10 cuvettes detects in the solution, record 10 kinds of tested amino acid whose spectrograms in 10 cuvettes, and the signal processing analysis comparison system (13) by described optical detection device of liquid porphyrin array, automatically its spectrogram and data are preserved and printout;
(3) analysis relatively reaches judgement
(2) step finish after, the signal processing analysis comparison system (13) of described optical detection device of liquid porphyrin array, automatically with the (2)-3) the tested amino acid whose spectrogram that gets of pacing, with preserve in the native system the (1)-4) the basic spectrogram of the known amino acid that gets of pacing analyzes comparison, judge tested amino acid whose kind, and with result's preservation and printout, and manually with the (2)-3) the tested amino acid whose spectrogram and the (1)-4 of step printout) the basic spectrogram that goes on foot printout analyzes comparison, also judges tested amino acid whose kind.
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